Investigations of extracellular matrix proteases, apoptotic and anti-apoptotic factors in the bovine corpus luteum

Kliem, Heike (2006): Investigations of extracellular matrix proteases, apoptotic and anti-apoptotic factors in the bovine corpus luteum. Dissertation, LMU München: Tierärztliche Fakultät

Abstract

The study is subdivided into two different parts: the first part deals with the development of a method to gain uterus milk in vivo during the preimplantation periode in cattle for the investigation of regulatory factors. The second part investigates different proteases in bovine follicles 20 hours after GnRH (Gonadotropin releasing hormone) injection (shortly bevor ovulation) for comparable as well as in the corpus luteum (CL) during oestrous cycle and induced luteolysis. In addition apoptotic as well as anti-apoptotic factors were evaluated in the CL during oestrous cycle and induced luteolysis. For the development of a method for gaining uterus milk in vivo during the first 24 days of gravidity in cattle, nine heifers were cycle synchronised using the Ovsynch method and artificially inseminated. Before flushing an epiduralanaesthesia was given and both uterus horns were flushed with 13ml 0.9% NaCl using a balloon embryo transfer catheter at day 5, 7, 12, 17 and 24 of gravidity. The catheter was placed 1cm cranial to the bifurcatio uteri in both horns. It was possible to retrive between 3ml and 13ml of the used flushing fluid. The uterus milk from the ipsilateral horn was inspected for an embryo and an EDTA-stabilisator was given to the uterus milk of both horns. An infection of the uterus occured in three heifers after the second and in five heifers after the third flushing. In one heifer no infection was found. Between day 17 and day 24 all heifers showed clear signs of oestrus. It was possible to detect progesterone, oestradiol-17-beta, PGF2alpha and VEGF via enzyme immunoassay (EIA) and radio immunoassay (RIA), respectively. Because of the occurred infection no statistic analysis was made. But it could be seen that the level of progesterone ranged between <0.1 and 0.4ng/ml, whereas no correlation to the inflammation was found. The values of the native uterus milk should be about 10-fold higher, because of the diluting effect of the flushing fluid. The pattern of oestradiol-17-beta was erratic, because the oestradiol levels of the flushing fluid from heifers with no infection till day 12 of gravidity could not be related to each other. It could be shown in further tests that high oestradiol-17-beta levels were measured via EIA in 0.9% NaCl with EDTA-stabilisator compared to 0.9% NaCl without the stabilisator. VEGF revealed levels between 0.08 and 2.58ng/ml, whereas the higher levels were seen during inflammation. The level of PGF2alpha showed a clear correlation to the infection. But there were also differences seen between the different heifers or even between the two uterus horns of one animal during the first flushing, which might be due to the massage of the uterus horns while flushing, which can induce prostaglandin secretion. The method of gaining uterus milk in vivo should be changed as follows: to prevent an endometritis just one flushing should be done per gravidity with gaining of the embryo, so that the heifer can be inseminated again after two oestrous cycle. Again both horns should be flushed, whereas one sterile disposable catheter should be used for each horn and the flushing should be devided into two parts: first flushing with 13ml of sterile 0.9% NaCL for biomolecular methodes, then flushing of the embryo using 5 x 50ml sterile PBS buffer. The embryo should be frozen in RNA-Later at -80° for further investigations. The received flushing fluid should be separated in two aliquotes, whereas one is used for the oestradiol-17-beta measurement and must not contain the EDTA-stabilisator. These two aliquotes should be centrifugated and the fluid should be stored seperatly at –20° from the received cell pellet. Part of the cell pellet can be used for the identification of the cells found in the uterus lumen and the rest stored in RNA-Later at –80° for further biomolecular investigations. For the evaluation of prostaglandins in the uterus milk it should be considered to treat the heifers with a prostaglandin synthetase inhibitor shortly before flushing. For the investigations of different extracellular matrix proteases and apoptotic as well as anti-apoptotic factors in bovine follicles and CL, RNA samples of a previous experiment were taken. In these experiment 26 cows received luteolytic dosis of a PGF2alpha-analogon and the CLs were removed by transvaginal ovaryectomy 0, 2, 4, 12, 24, 48 and 64 hours after induction of luteolysis. Additionally to these experiment five more cows were ovarectomised and the CLs were taken 0.5 hours after PGF2alpha treatment. For the investigation during oestrous cycle CL samples were collected at the slaughter house. For comparable reasons also follicles 20 hours after GnRH injection were taken using a superovulation model. The mRNA quality was inspected using the Bioanalyzer and good to very good results were seen for all samples. The mRNA expression levels were detected using quantitativ real time PCR (Rotor-Gene with SYBR Green I). The expression datas were normalised with the Bestkeeper index of four housekeeping genes using the delta delta CP method. The matrix metalloproteases (MMP-1, MMP-2, MMP-9, MMP-14, MMP-19), their tissue inhibitors (TIMP-1, TIMP-2), the plasminogen activator (PA) system (tissue –PA (tPA), urokinase-PA (uPA), urokinase-PA-receptor (uPAR), PA-inhibitor–1(PAI-1), PA-inhibitor-2 (PAI-2)), which are able degradate the extracellular matrix were investigated in follicles 20h after GnRH injection and in the CL during oestrous cycle and induced luteolysis. Furthermore the mRNA expression of the monocyte chemoattractant protein-1 (MCP-1), factors of the extrinsic apoptotic pathway (Tumor necrosis factor alpha (TNFalpha), TNF-Receptor-1 (TNFR1), TNF-Receptor-2 (TNFR2), Fas, Fas-Ligand (FasL)) and intrinsic apoptotic pathway (p53, Bax, Bcl-XL, Smac, Survivin) as well as the caspses 3, -6 and 7 were evaluated in the CL during oestrous cycle and induced luteolysis. During the first seven days of the oestrous cycle angiogenesis plays a critical role in the development of the CL. Basal membranes of blood vessels have to be removed to enable endothelial cell migration, which is necessary for the formation of new capillaries. MMP-1, MMP-2 and MMP-14 were not regulated, but TIMP-2, MMP-9, MMP-19 and TIMP-1 showed an increasing expression from 20h after GnRH till day 8-12 of the oestrous cycle in the CL. VEGF and bFGF seem to be able to stimulate the expression of MMP-1 and MMP-2 in luteal endothelial cells, which enable these cells to degrade the vascular basement membranes for sprouting new capillaries. MMP-19 is also expressed by these cells and has the same action like MMP-2 on basement membranes. The action of MMP-2 is not only regulated by VEGF and bFGF, but also by TIMP-2, which is lower expressed during angiogenesis in the CL and therefore might not block the action of MMP-2. TIMP-1 and TIMP-2 seem to be not only necessary for the inhibition of MMPs, but they are also able to enhance the proliferation of endothelial cell during angiogenesis. VEGF and bFGF also stimulate the expression of uPA in endothelial cells during angiogenesis. By forming a complex with uPAR and PAI-1 on the outer cell membrane these cells might be now able to detach from the vitronectin rich perivascular region and migrate towards the fibronection rich stroma of the CL in order to form new capillaries. Not only angiogenesis plays a critical role during CL formation, but also the inhibition of apoptotic action during vessel sprouting. Progesterone, bFGF and VEGF seem to inhibit an increased expression of apoptotic factors like p53 and Bax, while progesterone also inhibits Fas and caspase3. Addtitionally to the suppression of apoptotic factors an up-regulation of anti-apoptotic factors like Bcl-XL. and Survivin occurs. TNFalpha, produced by invading macrophages during CL formation, acts not only as an apoptotic factor, but is also able to enhance the production of PGE2 in endothelial and luteal cells, which stimulates together with TNFalpha the proliferation of endothelial cells during luteal angiogenesis. During induced luteolysis all investigated MMPs showed an increased expression as well as TIMP-1. Only TIMP-2 was down-regulated till 64h after PGF2alpha. PGF2alpha as luteolytic agents leads to the expression of MCP-1 on luteal endothelial cells. Thereupon monocytes and cytotoxic T cells migrate into the regressing CL and also start to express MCP-1 to recruite further mononuclear cells. For these migration into the stroma of the CL monocytes express uPA, uPAR and PAI-2. Macrophages are necessary for the phagocytosis of apoptotic cells and the degradation of ECM components in the CL during luteolysis. They are able to express different MMPs like MMP-1, MMP-9 and MMP-2. These MMPs degrade collagen I, the main structur collagen in the stroma of the CL, which might enhance the migration abilities of these macrophages. MMP-2 is also up-regulated in microvascular endothelial cells by TNFalpha, which is produced by macrophages. At the same time a down-regulation of TIMP-2 occurs, so that no inhibition of MMP-2 happens and a detachment of the endothelial cells from the vessel basement membrane follows. This leads to apoptosis of the endothelial cells, which seem to be the first cells to undergo cell death during luteolysis of the CL. Apoptosis seems to play an important role during luteolysis of the CL. The extrinsic pathway mediated by TNFalpha, its two receptors, Fas and FasL seem to be more important than the intrinsic pathway, due to the massiv up-regulation of these factors seen during induced luteolysis. TNFalpha and FasL show a high increased expression from the beginning of induced luteolysis onwards, which might by due to the infiltration of T cells and macrophages expressing these factors. Endothelial cells express TNFR1 and TNFR2 on their surface during luteolysis, which leads to the activation of the extrinsic apoptotic pathwas by binding of TNFalpha. INFgamma enhances these apoptotic action on endothelial cells. It seems possible that apoptosis of luteal cells occurs after the degradation of capillaries, which leads to an undersupply with oxygen and an increase of ROS. These reactive oxygen spieces up-regulate p53 and Bax as apoptotic factors of the intrinsic pathway, which leads to an activation of the caspases and apoptosis of luteal cells during induced luteolysis. These apoptotic action is further enhanced by the activation of the extrinsic pathway. Luteal cells stimulated by TNFalpha and INFgamma express Fas on their cell surface when the progesterone level declines. Cytotoxic T cells, express FasL and promote luteal cell death by activation the extrinsic pathway. This happens only when the progesterone level is decreased, which is known to happen at the end of the functional luteolysis. The results of these study show that ECM degrading proteases have two different roles in the life span of the CL: first they enable the sprouting of capillaries during angiogenesis in the developing CL, but during induced luteolysis their role seems to be to degrade ECM components of the CL stroma, which detaches endothelial cells from vessel basement membranes. These leads together with the infiltration of macrophages and cytotoxic T cells to apoptosis of endothelial and luteal cells.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: matrix metalloproteases, Apoptosis, Corpus Luteum, bovine
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 590 Tiere (Zoologie)
• Fakultäten: Tierärztliche Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 28. Juli 2006
• 1. Bericht­erstatter:in: Sinowatz, Fred